Data_Sheet_1_Reduction of Kcnt1 is therapeutic in mouse models of SCN1A and SCN8A epilepsy.docx

Developmental and epileptic encephalopathies (DEEs) are severe seizure disorders with inadequate treatment options. Gain- or loss-of-function mutations of neuronal ion channel genes, including potassium channels and voltage-gated sodium channels, are common causes of DEE. We previously demonstrated that reduced expression of the sodium channel gene Scn8a is therapeutic in mouse models of sodium and potassium channel mutations. In the current study, we tested whether reducing expression of the potassium channel gene Kcnt1 would be therapeutic in mice with mutation of the sodium channel genes Scn1a or Scn8a. A Kcnt1 antisense oligonucleotide (ASO) prolonged survival of both Scn1a and Scn8a mutant mice, suggesting a modulatory effect for KCNT1 on the balance between excitation and inhibition. The cation channel blocker quinidine was not effective in prolonging survival of the Scn8a mutant. Our results implicate KCNT1 as a therapeutic target for treatment of SCN1A and SCN8A epilepsy.</p

Pharmacometabolomic Signature of Ataxia SCA1 Mouse Model and Lithium Effects

<div><p>We have shown that lithium treatment improves motor coordination in a spinocerebellar ataxia type 1 (SCA1) disease mouse model (<i>Sca1^154Q/+</i>). To learn more about disease pathogenesis and molecular contributions to the neuroprotective effects of lithium, we investigated metabolomic profiles of cerebellar tissue and plasma from SCA1-model treated and untreated mice. Metabolomic analyses of wild-type and <i>Sca1^154Q/+</i> mice, with and without lithium treatment, were performed using gas chromatography time-of-flight mass spectrometry and BinBase mass spectral annotations. We detected 416 metabolites, of which 130 were identified. We observed specific metabolic perturbations in <i>Sca1^154Q/+</i> mice and major effects of lithium on metabolism, centrally and peripherally. Compared to wild-type, <i>Sca1^154Q/+</i> cerebella metabolic profile revealed changes in glucose, lipids, and metabolites of the tricarboxylic acid cycle and purines. Fewer metabolic differences were noted in <i>Sca1^154Q/+</i> mouse plasma versus wild-type. In both genotypes, the major lithium responses in cerebellum involved energy metabolism, purines, unsaturated free fatty acids, and aromatic and sulphur-containing amino acids. The largest metabolic difference with lithium was a 10-fold increase in ascorbate levels in wild-type cerebella (p<0.002), with lower threonate levels, a major ascorbate catabolite. In contrast, <i>Sca1^154Q/+</i> mice that received lithium showed no elevated cerebellar ascorbate levels. Our data emphasize that lithium regulates a variety of metabolic pathways, including purine, oxidative stress and energy production pathways. The purine metabolite level, reduced in the <i>Sca1^154Q/+</i> mice and restored upon lithium treatment, might relate to lithium neuroprotective properties.</p></div

Effect of Lithium treatment on blood plasma metabolic profile: Significantly different metabolites comparing Lithium treatment versus control conditions.

<p>Note: Bold indicates statistical significance. One-way ANOVA performed separately for wild-type and <i>Sca1^154Q/+</i> mice (see supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070610#pone-0070610-g002" target="_blank">Figure 2</a> for box-whisker plots). Abbreviation: Li, Lithium.</p

Supervised multivariate Partial Least Square separation of metabolic phenotypes.

<p><b>A.</b> Differences between plasma and cerebellum (vector v1) and between SCA1 and wild-type under control conditions (separated by vectors v3 and v4). <b>B.</b> Differences between Lithium treated and control conditions in cerebellum (vector v1) and between SCA1 and wild-type (separated by vectors v2 and v3). Abbreviations: Cereb, Cerebellum; Ctrl, Control; Li, Lithium; WT, Wild-type.</p

Effect of lithium treatment on cerebellum metabolome.

<p>Metabolic network of wild-type and <i>Sca1^154Q/+</i> cerebellum phenotypes. <b>A.</b> Wild-type mice. <b>B.</b> SCA1 knock-in mice. Red nodes: Increased metabolite levels under Lithium treatment; blue nodes: decreased levels. Node shades indicate ANOVA significance levels, node size reflect differences in magnitude of regulation. Red lines: reactant pair relationships obtained from the KEGG reaction pair database. Yellow solid lines: chemical similarity >0.5 Tanimoto score (Tanimoto scores range between 0 to 1, where 1 reflects identical structures). Yellow broken lines: chemically closest structure at <0.5 Tanimoto scores. Green circles group significant compounds that changed only in the Wild-type genotype. Orange circles group significant compounds that changed in both genotypes.</p

Genotype effect on metabolic profiles: Significantly different metabolites comparing wild-type versus <i>Sca1^154Q/+</i> mice under control conditions.

<p>Notes: Bold indicates statistical significance. One-way analysis of variance performed separately for cerebellum and blood plasma (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070610#pone-0070610-g002" target="_blank">Figure 2</a> and supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070610#pone-0070610-g001" target="_blank">Figure 1</a> for box-whisker plots). Abbreviation: NIST, National Institute of Standards and Technology.</p

Lithium treatment effect on blood plasma metabolome. Metabolic network of wild-type and <i>Sca1^154Q/+</i> plasma phenotypes.

<p><b>A.</b> Wild-type mice. <b>B.</b> SCA1 knock-in mice. Red nodes: Increased metabolite levels under Lithium treatment; blue nodes: decreased levels. Node shades indicate ANOVA significance levels, node size reflect differences in magnitude of regulation. Red lines: reactant pair relationships obtained from the KEGG reaction pair database. Yellow solid lines: chemical similarity >0.5 Tanimoto score (Tanimoto scores range between 0 to 1, where 1 reflects identical structures). Yellow broken lines: chemically closest structure at <0.5 Tanimoto scores.</p

Effect of introducing the <i>Sca1^154Q/+</i> gene into the wild-type genetic background for plasma and cerebellum.

<p>Individual box-whisker plots for selected significantly regulated metabolites. The whiskers encompass 1.5 of the interquartile range (IQR). Median value is indicated with a line in the box. The confidence diamonds indicate average values when the two samples are statistically different (colored boxplots, red for blood and grey for brain). Abbreviations: KI, SCA1 knock-in; WT, Wild-type.</p

Box-and-whisker plots: genotype-dependent metabolites in cerebellum tissue with significant differences between lithium and controls (<i>p</i><0.05).

<p><b>A.</b> Box-whisker plots for selected significantly regulated metabolites. <b>B.</b> Box-whisker plots for significantly regulated metabolites of purine metabolism pathway. The whiskers encompass 1.5 of the interquartile range (IQR). Median value is indicated with a line in the box. Boxes are filled in color (dark grey: SCA1 knock-in; light grey: wild-type) when the samples are statistically different between the two lithium treatments. Abbreviations: Ctl, Control; KI, SCA1 knock-in; Li, Lithium; WT, Wild-type.</p

Level of metabolites in SCA1 mice relative to wild-type non-treated mice: the treatment effect of lithium on impaired metabolites in SCA1 knock-in mice.

<p>Note: All values are relative to wild-type untreated animals. Abbreviations: Avg, Average; NIST, National Institute of Standards and Technology; SD, Standard deviation.</p